This invention relates generally to methods and compositions for use in recombinant DNA technology, particularly in methods for manipulation of DNA sequences encoding antibodies, proteins, or portions thereof.
The basic immunoglobulin (Ig) structural unit in vertebrate systems is composed of two identical xe2x80x9clightxe2x80x9d polypeptide chains (approximately 23 kDa), and two identical xe2x80x9cheavyxe2x80x9d chains (approximately 53 to 70 kDa). The four chains are joined by disulfide bonds in a xe2x80x9cYxe2x80x9d configuration, and the xe2x80x9ctailxe2x80x9d portions of the two heavy chains are bound by covalent disulfide linkages when the immunoglobulins are generated either by B cell hybridomas or other cell types.
A schematic of the general antibody structure is shown in FIG. 1. The light and heavy chains are each composed of a variable region at the N-terminal end, and a constant region at the C-terminal end. In the light chain, the variable region (termed xe2x80x9cVLJLxe2x80x9d) is composed of a variable (VL) region connected through the joining (JL) region to the constant region (CL). In the heavy chain, the variable region (VHDHJH) is composed of a variable (VH) region linked through a combination of the diversity (DH) region and the joining (JH) region to the constant region (CH). The VLJL and VHDHJH regions of the light and heavy chains, respectively, are associated at the tips of the Y to form the antibody""s antigen binding portion and determine antigen binding specificity.
The (CH) region defines the antibody""s isotype, i.e., its class or subclass. Antibodies of different isotypes differ significantly in their effector functions, such as the ability to activate complement, bind to specific receptors (e.g., Fc receptors) present on a wide variety of cell types, cross mucosal and placental barriers, and form polymers of the basic four-chain IgG molecule.
Antibodies are categorized into xe2x80x9cclassesxe2x80x9d according to the CH type utilized in the immunoglobulin molecule (IgM, IgG, IgD, IgE, or IgA). There are at least five types of CH genes (Cxcexc, Cxcex3, Cxcex4, Cxcex5, and Cxcex1), and some species (including humans) have multiple CH subtypes (e.g., Cxcex31, Cxcex32, Cxcex33, and Cxcex34 in humans). There are a total of nine CH genes in the haploid genome of humans, eight in mouse and rat, and several fewer in many other species. In contrast, there are normally only two types of light chain constant regions (CL), kappa (xcexa) and lambda (xcex), and only one of these constant regions is present in a single light chain protein (i.e., there is only one possible light chain constant region for every VLJL produced). Each heavy chain class can be associated with either of the light chain classes (e.g., a CHxcex3 region can be present in the same antibody as either a xcexa or xcex light chain), although the constant regions of the heavy and light chains within a particular class do not vary with antigen specificity (e.g., an IgG antibody always has a Cxcex3 heavy chain constant region regardless of the antibody""s antigen specificity).
Each of the V, D, J, and C regions of the heavy and light chains are encoded by distinct genomic sequences. Antibody diversity is generated by recombination between the different VH, DH, and JH gene segments in the heavy chain, and VL and JL gene segments in the light chain. The recombination of the different VH, DH, and JH genes is accomplished by DNA recombination during B cell differentiation. Briefly, the heavy chain sequence recombines first to generate a DHJH complex, and then a second recombinatorial event produces a VHDHJH complex. A functional heavy chain is produced upon transcription followed by splicing of the RNA transcript. Production of a functional heavy chain triggers recombination in the light chain sequences to produce a rearranged VLJL region which in turn forms a functional VLJLCL region, i.e., the functional light chain.
During the course of B cell differentiation, progeny of a single B cell can switch the expressed immunoglobulin isotype from IgM to IgG or other classes of immunoglobulin without changing the antigen specificity determined by the variable region. This phenomenon, known as immunoglobulin class-switching, is accompanied by DNA rearrangement that takes place between switch (S) regions located 5xe2x80x2 to each CH gene (except for Cxcex3) reviewed in Honjo (1983) Annu. Rev. Immunol. 1:499-528, and Shimizu and Honjo (1984) Cell 36:801-803). Sxe2x80x94S recombination brings the VHDHJH exon to the proximity of the CH gene to be expressed by deletion of intervening CH genes located on the same chromosome. The class-switching mechanism is directed by cytokines (Mills et al. (1995) J. Immunol. 155:3021-3036). Switch regions vary in size from 1 kb (Sxcex5) to 10 kb (Sxcex31), and are composed of tandem repeats that vary both in length and sequence (Gritzmacher (1989) Crit. Rev. Immunol. 9:173-200). Several switch regions have been characterized including the murine Sxcexc, Sxcex5, Sxcex1, Sxcex33, Sxcex31, Sxcex32b and Sxcex32a switch regions and the human Sxcexc switch region (Mills et al. (1995) supra; Nikaido et al. (1981) Nature 292:845-8; Marcu et al. (1982) Nature 298:87-89; Takahashi et al. (1982) Cell 29:671-9; Mills et al. (1990) Nucleic Acids Res. 18:7305-16; Nikaido et al. (1982) J. Biol. Chem. 257:7322-29; Stanton et al. (1982) Nucleic Acids Res. 10:5993-6006; Gritzmacher (1989) supra; Davis et al. (1980) Science 209:1360; Obata et al. (1981) Proc. Natl. Acad. Sci. U.S.A. 78:2437-41; Kataoka et al. (1981) Cell 23:357; Mowatt et al. (1986) J. Immunol. 136:2674-83; Szurek et al. (1985) J. Immunol. 135:620-6; and Wu et al. (1984) EMBO J. 3:2033-40).
Observations that a single B cell can express more than one isotype simultaneously on its surface is not explained by the class-switching mechanism since Sxe2x80x94S recombination is limited to intrachromosomal recombination and results in deletion of the exchanged CH gene. A second mechanism, called trans-splicing, has been described in which two transcripts generated from different chromosomes are joined to form a single continuous transcript (Shimizu et al. (1991) J. Exp. Med. 173:1385-1393). Transgenic mice carrying a rearranged expressible VHDHJH heavy chain xcexc gene integrated outside the mouse IgH locus were found to produce mRNA having the VHDHJH region of the transgene correctly spliced to the endogenous CH region. As with Sxe2x80x94S recombination, the frequency of trans-splicing is low, and the factors regulating both mechanisms are not well understood.
The value and potential of antibodies as diagnostic and therapeutic reagents has been long-recognized in the art. Unfortunately, the field has been hampered by the slow, tedious processes required to produce large quantities of an antibody of a desired specificity. The classical cell fusion techniques allowed for efficient production of monoclonal antibodies by fusing the B cell producing the antibody with an immortalized cell line. The resulting cell line is called a hybridoma cell line. However, most of these monoclonal antibodies are produced in murine systems and are recognized as xe2x80x9cforeignxe2x80x9d proteins by the human immune system. Thus the patient""s immune system elicits a response against the antibodies, which results in antibody neutralization and clearance, and/or potentially serious side-effects associated with the anti-antibody immune response.
One approach to this problem has been to develop human or xe2x80x9chumanizedxe2x80x9d monoclonal antibodies, which are not as easily xe2x80x9crecognizedxe2x80x9d as foreign epitopes, and avoid an anti-antibody immune response in the patient. Applications of human B cell hybridoma-produced monoclonal antibodies have promising potential in the treatment of cancer, microbial, ad viral infections, B cell immunodeficiencies associated with abnormally low antibody production, autoimmune diseases, inflammation, transplant rejection and other disorders of the immune system, and other diseases. However, several obstacles remain in the development of such human monoclonal antibodies. For example, many human tumor antigens may not be immunogenic in humans and thus it may be difficult to isolate human B cells producing antibodies against human antigens.
Attempts to address the problems associated with antibodies for human therapeutics have used recombinant DNA techniques. Most of these efforts have focused on the production of chimeric antibodies having a human CH region and non-human (e.g., murine) antigen combining (variable) regions. These chimeric antibodies are generally produced by cloning the desired antibody variable region and/or constant region, combining the cloned sequences into a single construct encoding all or a portion of a functional chimeric antibody having the desired variable and constant regions, introducing the construct into a cell capable of expressing antibodies, and selecting cells that stably express the chimeric antibody. Alternatively, the chimeric antibody is produced by cloning the desired variable region or constant region, introducing the construct into an antibody-producing cell, and selecting for chimeric antibody-producing cells that result from homologous recombination between the desired variable region and the endogenous variable region, or the desired constant region and the endogenous constant region. Examples of techniques which rely upon recombinant DNA techniques such as those described above to produce chimeric antibodies are described in PCT Publication No. WO 86/01533 (Neuberger et al.), and in U.S. Pat. No. 4,816,567 (Cabilly et al.) and U.S. Pat. No. 5,202,238 (Fell et al.). These methods require transferring DNA from one cell to another, thus removing it from its natural locus, and thus require careful in vitro manipulation of the DNA to ensure that the final antibody-encoding construct is functional (e.g., is capable of transcription and translation of the desired gene product).
There is a clear need in the field for a method for producing a desired protein or antibody which does not require multiple cloning steps, in more efficient than conventional homologous recombination, and can be carried out in hybridoma cells.
The present invention features a method of replacing one DNA sequence with another using switch (S) regions derived from an immunoglobulin (Ig) gene. The method of the invention allows any two pieces of DNA to be xe2x80x9cswitchedxe2x80x9d or a piece of exogenous DNA to be inserted into a site containing a natural or artificial S region. Thus the method of the invention allows directed recombination to occur and eliminates many cloning steps required by current recombinant DNA methods.
In the method of the invention, directed recombination is brought about between a targeting construct and a target locus. The nucleic acid targeting construct is composed minimally of a switch region and a promoter operably linked to and 5xe2x80x2 of the switch region. Additionally, depending on the desired recombinatorial product, the targeting construct can also contain a modifying sequence operably linked to and 3xe2x80x2 of the switch region, and other DNA sequences between the promoter and switch regions, e.g., 5xe2x80x2 of the switch region and 3xe2x80x2 of the promoter region. Of particular interest is the use of a targeting construct with an Ig heavy chain to facilitate isotype switching, e.g., replacement of an endogenous constant region (CH) in an antibody heavy chain gene (target sequence) with a CH of a different subtype, isotype, or species of origin (modifying sequence). For example, exogenous DNA encoding the constant or variable region of an antibody light or heavy chain can be switched with the constant or variable region of an endogenous sequence to create a sequence which encodes an antibody with a different constant or variable region. In a broader sense, the method of the invention is widely applicable to manipulate DNA sequences for production of a desired protein or protein component, including the production of chimeric antibodies having a desired variable region linked to a non-antibody polypeptide (e.g., a detectable polypeptide label, or a polypeptide having a desired activity).
In one aspect, the invention features a method for directed switch-mediated recombination by a) introducing a targeting construct into a cell having a target locus, the target locus being minimally composed of a promoter, a switch region, and a target sequence, wherein the targeting construct is minimally composed of a promoter and a switch region, and can contain additional modifying sequences, b) culturing the cell to allow transcription of the target locus and the targeting construct, thereby promoting recombination of the switch regions of the target locus and the targeting construct, and c) selecting a cell containing the desired recombined DNA product sequence, minimally composed of a switch region (composed of DNA sequences from one or both the target locus switch region and targeting construct switch region).
In a specific embodiment of the invention, the targeting construct (P1-S1) is composed of a promoter (P1) and switch region (S1) and the target locus (P2-S2-T) is composed of a promoter (P2), a naturally occurring or artificially inserted switch region (S2), and a target sequence (T). Directed Sxe2x80x94S recombination between the Sxe2x80x94S regions results in a DNA sequence having the P1 promoter of the targeting construct, a switch region containing DNA sequences from one or both S1 and S2 regions, and the T sequence (P1-S1/S2-T). In this embodiment, the target sequence is removed from the control of the target locus promoter and placed under control of the desired P1 promoter. Cells containing the desired DNA sequence are selected by methods known in the art, including Southern blot analysis or PCR.
In another embodiment, the targeting construct (P1-S1-M) is composed of a promoter (P1), a switch region (S1), and a modifying sequence (M), and the target locus (P2-S2-T) is composed of a promoter (P2), a naturally occurring or artificially inserted switch region (S2), and a target sequence (T). Directed Sxe2x80x94S recombination between the Sxe2x80x94S regions results in two possible recombinatorial product sequences, one having the P1 promoter of the targeting construct, a switch region containing DNA sequences from one or both S1 and S2 regions, and the T sequence (P1-S1/S2-T), and a second sequence having a P2 promoter, a switch region containing DNA sequences from one or both S1 and S2 regions, and the M sequence (P1-S1/S2-M). In this embodiment, cells expressing the M sequence are selected by methods known in the art, including Southern or Northern blot analysis.
In a third embodiment, the targeting construct (P1-Z-S1) is composed of a promoter (P1), DNA sequences 5xe2x80x2 to the switch region (Z), and the switch region (S1). The target locus (P2-S2-T) is composed of a promoter (P2), a naturally occurring or artificially inserted switch region (S2), and a target sequence (T). Directed Sxe2x80x94S recombination between the switch regions results in a DNA sequence having the P1 promoter of the targeting construct, the Z DNA sequences, a switch region containing DNA sequences from one or both switch regions, and the T sequence (P1-Z-S1/S2-T).
The target locus is a DNA sequence having a switch region, and may be a native, naturally-occurring sequence (e.g., an Ig locus of an antibody-producing cell), a rearranged Ig locus, or a recombinantly produced DNA sequence artificially inserted at a desired site. The target locus can be either an extrachromosomal element or a stably integrated chromosomal element. Preferably, the target locus encodes an antibody heavy chain gene. The targeting construct is either an extrachromosomal element or a stably integrated chromosomal element. Where the target locus is an antibody heavy chain gene, the modifying sequence of the targeting construct preferably encodes a different or modified heavy chain constant region or a non-antibody sequence of interest (e.g., a detectable polypeptide label, an enzyme, a toxin, or a growth factor).
The invention provides a method of modifying a DNA sequence by directed Sxe2x80x94S recombination. The invention allows DNA recombination to be directed to any site which contains a naturally-occurring switch region or synthetic switch region, including a site into which an S region has been artificially inserted.
The invention provides a method to replace or modify a first DNA sequence (a target sequence) with a second DNA sequence (a modifying sequence) without the need for isolating the nucleotide sequence containing the target sequence, excising the target sequence, and ligating the modifying sequence in place of the target sequence. The invention also provides a method to replace portions of a polypeptide-encoding sequences with a heterologous amino acid sequence, where the polypeptide is composed of two distinct components (e.g., an N-terminal component and a C-terminal component) that, for example, confer distinct functional or structural characteristics upon the polypeptide (e.g., ligand binding or cell-binding). For example, the invention allows for the substitution of either the N-terminal portion with a different, heterologous amino acid-encoding sequence, or the C-terminal portion with a different, heterologous amino acid-encoding sequence.
Directed switch-mediated recombination allows recombination to occur at a specific, pre-selected region with an increased efficiency relative to the naturally-occurring mechanism which is limited to the immunoglobulin heavy chain. The method of the invention removes switch-mediated recombination from the limitations of its normal regulatory environment, allowing recombination to be controlled as needed with, for example, the use of constitutive or inducible promoters.
The ability to accomplish directed in vitro S-mediated recombination avoids tedious, time-consuming manipulation of DNA using conventional recombinant DNA techniques while providing a highly efficient method of inserting a DNA sequence. For example, the method allows the detectable label portion of fusion proteins (e.g., xcex2-galactosidase) to be readily exchanged for a different amino acid sequence (e.g., alkaline phosphatase).
In a specific application of the method of the invention, directed Sxe2x80x94S recombination is used to replace the constant region of an antibody heavy chain gene with a different or modified constant region without the need for extensive manipulation of the antibody heavy chain gene. Additionally, the method of the invention allows the antibody gene to be maintained in its native locus.
These and other objects, advantages and features of the present invention will become apparent to those persons skilled in the art upon reading the details of the compositions, composition components, methods and method steps of the invention as set forth below.